Bottom Line:
By combining these methods, a high heterogeneity of DENV N-glycans was found.Five types of N-glycan were identified on DENV-2, including mannose, GalNAc, GlcNAc, fucose and sialic acid; high mannose-type N-linked oligosaccharides and the galactosylation of N-glycans were the major structures that were found.For the first time, this study provides a comprehensive understanding of the N-linked glycan profile of whole DENV-2 particles derived from insect cells.

ABSTRACTDENV envelope glycoprotein (E) is responsible for interacting with host cell receptors and is the main target for the development of a dengue vaccine based on an induction of neutralizing antibodies. It is well known that DENV E glycoprotein has two potential N-linked glycosylation sites at Asn67 and Asn153. The N-glycans of E glycoprotein have been shown to influence the proper folding of the protein, its cellular localization, its interactions with receptors and its immunogenicity. However, the precise structures of the N-glycans that are attached to E glycoprotein remain elusive, although the crystal structure of DENV E has been determined. This study characterized the structures of envelope protein N-linked glycans on mature DENV-2 particles derived from insect cells via an integrated method that used both lectin microarray and MALDI-TOF-MS. By combining these methods, a high heterogeneity of DENV N-glycans was found. Five types of N-glycan were identified on DENV-2, including mannose, GalNAc, GlcNAc, fucose and sialic acid; high mannose-type N-linked oligosaccharides and the galactosylation of N-glycans were the major structures that were found. Furthermore, a complex between a glycan on DENV and the carbohydrate recognition domain (CRD) of DC-SIGN was mimicked with computational docking experiments. For the first time, this study provides a comprehensive understanding of the N-linked glycan profile of whole DENV-2 particles derived from insect cells.

pone.0132122.g011: The proposed structural information of glycan on the surface of mature DENV-2 revealed by Mass Spectrometry and lectin microarray analysis.

Mentions:
It is known that lectin array has an advantage of obtaining detailed information about the structures of partial glycans, especially their linkage information. In addition, mass spectrometry is usually utilized to obtain information about the compositions of oligosaccharides[25]. To acquire precise structural information about the glycan profile of DENV, lectin array and mass spectrometry were employed in tandem in this study. When integrating the analyses of these results, precise structural information of the glycans on the surface of mature DENV-2 was obtained, which is shown in Fig 11. Consistent with a previous report, a high heterogeneity was found in the N-glycans on DENV [26]. Five types of N-glycans were identified on DENV-2, including mannose, GalNAc, GlcNAc, fucose and sialic acid, and high-mannose-type N-linked oligosaccharides and galactosylation of N-glycan were the major structures. According to the results of lectin arrays, the mannose branching/high-mannose-type N-glycan binders GNA, HHL, NPA and ConA showed positive binding signals, which indicated that mannose branching and high-mannose-type N-glycans exist in DENV. As a result of MS/MS, the fragment ions B3Y3β (671.201) and B4αY4β(1095.370)at m/z 1419.476 indicated the presence of oligosaccharides with mannose branches; furthermore, an MS/MS spectrum of a glycan at m/z 1419.476 revealed that high-mannose-type N-glycans exist in DENV. MS/MS analysis revealed ionsC5Y5β(1298.449) and Y5β(1501.529) at m/z 1663.581 andC4Y5α (1460.502)and Y5α(1866.661) at m/z 2028.714, which were indicative of the presence of terminal GalNAc. Meanwhile, the binding signals of the gal binders ACA, RCA120, EEL, BS-I and BPL on lectin arrays also confirmed the existence of gal residues. The lectins PHA-E and PHA-E+L were detected via moderate binding signals, which revealed that bisecting GlcNAc, biantennary or tetraantennary complex-type N-glycans existed in the glycome of DENV. The ions at m/z 2654.840 and 2983.878 exhibited bisecting GlcNAc residues, and the predicted structures at m/z 1663.581, 2028.648 and 2983.878 in the MS spectrum verified the existence of biantennary or tetraantennary complex-type N-glycans on DENV. The results of MS also detected an N-glycan on DENV with an internal fucose residue (at m/z 1809.517, 2133.615, 2174.623,etc.) and an external fucose residue that was attached to GlcNAc (at m/z 2289.816, 2654.840 and 2983.878). Furthermore, according to the glycans recognized by the lectins AAL, PSA and LCA, it was indicated that α-fucose, especially α1,6-fucose (a core fucose that is recognized by PSA and LCA),was present on DENV. A sialic acid N-glycan was speculated to be present by MS spectra at m/z 2289.816, 2341.702, 2654.840 and 2983.878. Sialylation of DENV glycans was also identified by lectin array according to the binding signals of the lectins MAL-II (Siaα2,3-Gal binder), SNA (Siaα2,6-Gal binder) and WGA (multivalent sialic acid binder). It was noted that the intensity of the binding signal corresponding to SNA was almost twice that of MAL-II. This indicated the presence of sialic acids in DENV glycans. Moreover, the abundance of Siaα2,6-Gal was higher than Siaα2,3-Gal.

pone.0132122.g011: The proposed structural information of glycan on the surface of mature DENV-2 revealed by Mass Spectrometry and lectin microarray analysis.

Mentions:
It is known that lectin array has an advantage of obtaining detailed information about the structures of partial glycans, especially their linkage information. In addition, mass spectrometry is usually utilized to obtain information about the compositions of oligosaccharides[25]. To acquire precise structural information about the glycan profile of DENV, lectin array and mass spectrometry were employed in tandem in this study. When integrating the analyses of these results, precise structural information of the glycans on the surface of mature DENV-2 was obtained, which is shown in Fig 11. Consistent with a previous report, a high heterogeneity was found in the N-glycans on DENV [26]. Five types of N-glycans were identified on DENV-2, including mannose, GalNAc, GlcNAc, fucose and sialic acid, and high-mannose-type N-linked oligosaccharides and galactosylation of N-glycan were the major structures. According to the results of lectin arrays, the mannose branching/high-mannose-type N-glycan binders GNA, HHL, NPA and ConA showed positive binding signals, which indicated that mannose branching and high-mannose-type N-glycans exist in DENV. As a result of MS/MS, the fragment ions B3Y3β (671.201) and B4αY4β(1095.370)at m/z 1419.476 indicated the presence of oligosaccharides with mannose branches; furthermore, an MS/MS spectrum of a glycan at m/z 1419.476 revealed that high-mannose-type N-glycans exist in DENV. MS/MS analysis revealed ionsC5Y5β(1298.449) and Y5β(1501.529) at m/z 1663.581 andC4Y5α (1460.502)and Y5α(1866.661) at m/z 2028.714, which were indicative of the presence of terminal GalNAc. Meanwhile, the binding signals of the gal binders ACA, RCA120, EEL, BS-I and BPL on lectin arrays also confirmed the existence of gal residues. The lectins PHA-E and PHA-E+L were detected via moderate binding signals, which revealed that bisecting GlcNAc, biantennary or tetraantennary complex-type N-glycans existed in the glycome of DENV. The ions at m/z 2654.840 and 2983.878 exhibited bisecting GlcNAc residues, and the predicted structures at m/z 1663.581, 2028.648 and 2983.878 in the MS spectrum verified the existence of biantennary or tetraantennary complex-type N-glycans on DENV. The results of MS also detected an N-glycan on DENV with an internal fucose residue (at m/z 1809.517, 2133.615, 2174.623,etc.) and an external fucose residue that was attached to GlcNAc (at m/z 2289.816, 2654.840 and 2983.878). Furthermore, according to the glycans recognized by the lectins AAL, PSA and LCA, it was indicated that α-fucose, especially α1,6-fucose (a core fucose that is recognized by PSA and LCA),was present on DENV. A sialic acid N-glycan was speculated to be present by MS spectra at m/z 2289.816, 2341.702, 2654.840 and 2983.878. Sialylation of DENV glycans was also identified by lectin array according to the binding signals of the lectins MAL-II (Siaα2,3-Gal binder), SNA (Siaα2,6-Gal binder) and WGA (multivalent sialic acid binder). It was noted that the intensity of the binding signal corresponding to SNA was almost twice that of MAL-II. This indicated the presence of sialic acids in DENV glycans. Moreover, the abundance of Siaα2,6-Gal was higher than Siaα2,3-Gal.

Bottom Line:
By combining these methods, a high heterogeneity of DENV N-glycans was found.Five types of N-glycan were identified on DENV-2, including mannose, GalNAc, GlcNAc, fucose and sialic acid; high mannose-type N-linked oligosaccharides and the galactosylation of N-glycans were the major structures that were found.For the first time, this study provides a comprehensive understanding of the N-linked glycan profile of whole DENV-2 particles derived from insect cells.

ABSTRACTDENV envelope glycoprotein (E) is responsible for interacting with host cell receptors and is the main target for the development of a dengue vaccine based on an induction of neutralizing antibodies. It is well known that DENV E glycoprotein has two potential N-linked glycosylation sites at Asn67 and Asn153. The N-glycans of E glycoprotein have been shown to influence the proper folding of the protein, its cellular localization, its interactions with receptors and its immunogenicity. However, the precise structures of the N-glycans that are attached to E glycoprotein remain elusive, although the crystal structure of DENV E has been determined. This study characterized the structures of envelope protein N-linked glycans on mature DENV-2 particles derived from insect cells via an integrated method that used both lectin microarray and MALDI-TOF-MS. By combining these methods, a high heterogeneity of DENV N-glycans was found. Five types of N-glycan were identified on DENV-2, including mannose, GalNAc, GlcNAc, fucose and sialic acid; high mannose-type N-linked oligosaccharides and the galactosylation of N-glycans were the major structures that were found. Furthermore, a complex between a glycan on DENV and the carbohydrate recognition domain (CRD) of DC-SIGN was mimicked with computational docking experiments. For the first time, this study provides a comprehensive understanding of the N-linked glycan profile of whole DENV-2 particles derived from insect cells.